Type I diabetes (TID) patients suffer from insufficient or lack of insulin to regulate blood glucose levels. Due to loss of pancreatic beta-cells, the only treatment for TID patient is transplantation of donor islets. With the improved transplantation technique, it still requires 2-4 donor pancreas for each successful transplantation. The shortage of donor islets and inefficiency of beta-cell regeneration presented an urgent need for alternative sources of beta-cells. Embryonic stem (ES) cell-derived insulin producing cells are regarded as a possible means to overcome these limitations. However, difficulties in controlling the fate of ES cell differentiation have presented significant challenge for using ES cells to treat type I diabetes. Current protocols of ES cell differentiation to beta-cells rely on trying different combinations of medium factors. A lack of monitoring system has made the effort to improve the beta-cell proliferation protocol a blind search. In this grant proposal, we intend to design a monitoring system to monitor the differentiation process. This monitoring system is based on the physiological development of the pancreas. During pancreas development, several distinct stage with different cell markers have been identified to be critical. Using these markers as a monitoring guide will aid in our effort of improving beta-cell differentiation protocol. In this study, we propose to design fluorescent reporter markers to monitor the appearance and disappearance of these markers. The system designed here will be useful tools for future studies targeted at improving the current beta-cell differentiation protocol. A efficient and reliable beta-cell differentiation protocol is the critical step toward using ES cell based therapy for the treatment of type I diabtetes.
Statement of Benefit to California:
The proposed study is an important step towards establishing a reliable, robust differentiation system for beta-cells. The percentage of population who has diabetes in California is among the top in the 50 states. Therefore, a treatment targeted at diabetes will be of significant benefit to the California citizens. For type I diabetes for whom transplantation is the only reliable treatment, an alternative treatment is urgently needed and is of significant benefit to the Californians.
SYNOPSIS: The goal of the proposed research is to create a multi-membered genetic reporter system in hESCs useful to monitor the directed differentiation of cultured ESCs toward the pancreatic beta-cell phenotype. Toward this goal, Aim 1 will derive separate hESC lines with GFP, YFP or RFP reporters for the expression of the Sox17, Pdx1, Ngn3 & Nkx2.2 genes (plus insulin), which not only mark but also control pancreatic endocrine cell development. Three strategies for homologous recombination will be attempted in order until one is successful. Aim 2 will test the veracity of expression of the reporters in 3 ways: a, by the parallel insertion of similar constructs in the orthologous genes in mouse ES cells and analysis in embryos; b, by the creation of teratomas in mice with hESC reporter lines and correlation with endogenous pancreatic endocrine markers; and c, testing whether reporters coexpress with panreatic markers during induction in vitro, by methods effective for mouse ESCs. SIGNIFICANCE AND INNOVATION: The generation of replacement beta-cells for transplantation therapy of diabetics is a major and highly worthy goal of stem cell technology. Progress has been limited for hESCs, in part due to lack of cell lines and reagents as are available for the equivalent mouse work. Engineered human stem cell lines with intrinsic reporter genes that can be used to monitor separate endocrine developmental stages during attempts to induce step-wise beta-cell differentiation in vitro would be an important technical advance. The proposed research will use state-of-the-art technologies to derive useful hESC lines. The PI proposes to create and test reporter hESC lines for endodermal and pancreatic differentiation. She will create several fluorescently tagged reporter gene constructs, introduce these into mouse and hESCs, and test the constructs in vitro and in vivo. The approach is innovative and the research is potentially very significant. STRENGTHS: A strength of this proposal is the plan to explore several recombination strategies to create intrinsic FP reporters in hESCs that would precisely mimic the expression of definitive markers for embryonic beta-cell development, from endoderm to the mature beta-cell. Although homologous recombination experiments with hESCs have been difficult, the recruitment of Dr. Martin Pera, who has extensive pioneering success with this strategy and is Director of the Center for Stem Cell and Regenerative Medicine in the Broad Institute at USC, greatly enhances the chance for success. A limitation of creating reporter lines in this manner is the lack of a direct assay for proper developmental expression of the engineered reporters. To overcome this, three different assays are proposed: a) first, engineering equivalent reporters in mouse ESCs followed by generating embryos to verify correct spatial and temporal expression; second, analysis by the creation of teratomas in mice with hESC reporter lines and correlation of expression with pancreatic endocrine markers; and third, testing whether the reporters co-express with pancreatic markers during induction of hESC differentiation in vitro using protocols known to be effective for mouse ESCs. There are assays to follow the progress of hESC differentiation in vitro, such as real-time RT-PCR and immunolocalization, other than using an intrinsic fluorescent reporter. But these do not have the advantage of being able to both assess the fraction of cells in the population at a particular developmental stage and use the potential for FACS enrichment of these, potentially minority cell populations, for next-step induction experiments. The PI has good experience creating similar constructs to those proposed here. She has worked with mouse ESCs and created transgenic mice. The Preliminary data presented demonstrates that some of this work has begun in mouse ESCs. There is a high probability of success using only mouse ESCs. The PI recognizes the difference between mouse and human ESCs and realizes that there are substantial hurdles to be overcome in order to carry out the same work using hESC lines. The first Specific Aim of this proposal is to create fluorescent constructs using GFP, YFP and RFP expressed off of the Sox17, pdx-1, ngn-3, Nkx2.2, and insulin promoters, using homologous recombination. This is not a trivial task. There are very few reports of successful attempts of homologous recombination in hESC lines and this proposal seems very ambitious in its aims. The PI will first test the constructs in mouse ESCs. The validity of this is in question. There are a few significant differences between mouse and human ESCs and showing efficacy in mouse ESCs does not guarantee success in hESCs. The work will be carried out at USC and has the support of the USC Stem Cell Center. This is significant and interaction with the Center will benefit the project since the PI and her team do not have much hESC experience. WEAKNESSES: Each of the assays proposed to verify correct reporting by the fluorescent proteins has intrinsic limitations. a) Creating equivalent reporters in mouse embryos is time consuming, involves recreation of targeting vectors for homologous recombination with mouse gene sequences, is sensitive to any mouse-specific problems with the locus, and is insufficient alone and would require analysis of the human reporters in a human background. b) Analyses of human teratomas generated in mice with hESC reporter lines does not provide answers to important questions about timing of developmental expression and is complicated by the lack of specificity of many reporters and markers between visceral and definitive endoderm. c) Using the same in vitro induction assay of ESC differentiation to verify the accuracy of reporter gene expression that will later use the reporter to follow endocrine differentiation does not provide a necessary independent test. If the verification is inaccurate, then that assay will direct the experimental results to that inaccurate end. These problems need careful consideration in advance. Some of the markers that will be used to follow development have complexities that have not been considered. Sox17 and Pdx1 markers are also detected in visceral endoderm in mice and this may confound some differentiation experiments if this expression pattern extends to human embryos and is recapitulated during induced development in culture. During pancreatic development, Nkx2.2 is expressed early in precursor epithelium and then late in differentiating beta-cells, and so is an ambiguous marker for endocrine cell development. Other useful markers should be considered: Ptf1a is more specific than Pdx1 for pancreatic endoderm; Hnf1-beta and Sox9 are key markers for pre-Ngn3-committed pancreatic epithelial cells; and a most useful beta-cell marker is MafA, the first key and specific regulator of beta-cell differentiation. The PI consider Ptf1a, Hnf1-beta, Sox9 and MafA as appropriate alternative markers for following hESC differentiation. Considering the complexities of homologous recombination in hESCs, the testing protocols and the pace of animal work, the timeline of 2 years is unrealistically short. As written, this proposal is not eligible for NIH funding because of the hESC lines chosen - 2 of the Harvard-HHMI HUES cell lines. The only mention of why these lines were chosen appears on page 2 of the Research Proposal where the PI states that they require “hES cells that have not been grown on mouse feeders.” However, the HUES lines were derived and expanded on mouse feeders, very similar to the NIH-eligible hESC lines. The large part of this work that deals with mouse ESC lines is eligible for NIH funding. Feasibility: The PI only designates 5% of her time to this project. Her role is stated as being responsible for all experimental planning, data analysis and distribution, writing manuscripts, and training and directing technical staff. It is hard to imagine how all this will be accomplished with only a 5% commitment. A few minor problems were found with this proposal. In the Oversight section – this proposal does NOT use human oocytes, human in-vitro embryos, or derive a covered stem cell line. This project does NOT require IRB approval, nor does it involve the use of human subjects. However, the project does use hESC lines and these cells should be considered biohazardous materials as they are human tissue. There were also a few issues with the Budget: the amount budgeted for travel seems high and the PI requested 3 pieces of equipment very close to $5000 each – electroporator, spectrophotometer, and microscope upgrade. The equipment needed to perform the work should be in place at the time of award (also goes to feasibility). DISCUSSION: There was no further discussion beyond the comments by the reviewers